African Journal of Biotechnology Vol. 9(34), pp.

5611-5615, 23 August, 2010

Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2010 Academic Journals

Full Length Research Paper

Evaluation of Viburnum foetens for anticancer and

antibacterial potential and phytochemical analysis
Yamin Bibi1, Sobia Nisa1, Abdul Waheed2, Muhammad Zia3*, Sadia Sarwar1, Sabbir Ahmed4
and M. Fayyaz Chaudhary1
1
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan.
2
School of Pharmacy and Chemistry, Kingston University, UK.
3
Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan.
4
School of Science, Faculty of Science and Technology, University of the West of Scotland, UK.
Accepted 8 July, 2010

Viburnum foetens is an ethanobotanically important plant species traditionally used as purgative and
also have sedative properties. Methanol extract from leaf explants was used to determine cytotoxic and
antibacterial potential of this potent shrub. It was observed that cytotoxicity against MCF-7 cell lines
gradually increases according to concentration of extract used. Further, the methanol crude extract was
fractionated on polarity basis and each fraction was again tested for cytotoxic potential. Methanol
fraction showed 83% inhibition that was highest from all other fractions. The crude methanol extract
showed mild activity ranging from 10.30 to 12.00 mm zones of inhibition against bacterial strains tested
by agar well diffusion method. Phytochemical analysis reveals that methanol fraction contain
flavonoids, coumarins and tannins only, while crude extract include other phytochemicals along with
these. It can be suggested that V. foetens contains some anticancerous compounds to be isolated and
can be used as drug.

Key words: Anticancer, cytotoxic, MCF-7 cell line, MTT assay, phytochemical, Viburnum foetens.

INTRODUCTION

Plants have been used for medicinal purposes 60,000
years ago (Solecki and Shanidar, 1975). According to an
analysis by World Health Organization, nearly 80% of the
world population depends on herbal medicines for their
health care problems (Farnsworth et al., 1985). The
medicinal use of plants is actually due to the presence of
active components that are effective for human body in
many ways (Akinmoladun et al., 2007). Flavonoids,
alkaloids, essential oils, tannins, terpenoids, saponins
and phenolic compounds are some of the constituents
responsible for bioactivity of plants (Edeoga et al., 2005;
Tan et al., 2006). Synthesis of such compounds is one of
the mechanisms responsible for the ability of plants to
*Corresponding author. E-mail: ziachaudhary@gmail.com. Tel:
+92-51-90643011.
Abbreviations: DMEM, Dulbecco's modified eagle medium;
DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
resist themselves from predators as well as to destroy
pathogenic microorganisms and unwanted cells (Mans et
al., 2000).
Among the medicinal plants, about one thousand plant
species were found to have anticancer potential out of
250,000 plant species existing on earth (Mukherjee et al.,
2001). Likewise, plant extracts exhibiting antimicrobial
activity are a good source of antimicrobial compounds
and hence antibiotic in nature (Cowann, 1999). Bioactivity
guided isolation technique is the key to the discovery of
some important anticancer agents like paclitaxel from
Taxus brevifolia and camptothecin from Camptotheca
acuminate (Kinghorn, 1994).
Genus Viburnum belonging to family Adoxaceae consists
of about 200 species distributed from South America to
South East Asia (Lobstein et al., 1999). Traditionally
Viburnum species have been used in medicines due to
their diuretic, antispasmodic and sedative effects (Cometa
et al., 1998). Flavonoids, biflavonoids and coumarins are
reported in different Viburnum species (Glasby, 1991;
Plouvier, 1992). Hepatoprotective, antioxidant, antinoci-
5612 Afr. J. Biotechnol.
ceptive, anti inflammatory and gastroduodeno-protective
activities have been studied in genus Viburnum (Kim et
al., 2003; Mohamed et al., 2005; Yılmaz et al., 2007;
Zayachkivska et al., 2006).
Viburnum foetens is a large deciduous shrub. It is
widely distributed in Himalaya at an altitude between
1500 -3000 m from Swat eastward to Bhutan, South Tibet
(Flora of Pakistan). Fruit of plant is edible and leaves
have foetid aroma (Hedrick, 1972; Tanaka, 1976).
Ethanobotanical survey shows that plant has been used
traditionally as purgative and has sedative properties.
Branches have been used by local people as tooth brush
(miswaak) for cleaning teeth (Qureshi et al., 2009).
Antidiabetic activity of the V. foetens has been studied
(Hussain et al., 2003).
To the best of our knowledge, no other biological activity
as well as phytochemical investigation of this plant has
been done so far. The present investigation aims to do
the preliminary phytochemical analysis and to evaluate
potential of plant extract against breast cancer cell line as
well as against gram negative and gram positive bacterial
strains.
MATERIALS AND METHODS
Collection and identification of plant material
Fresh plant material was collected from northern areas of Pakistan
(Ayubia NWFP Pakistan). Plant was identified by a Taxonomist,
Department of Plant Sciences, Quaid-i-Azam University Islamabad.
Drying and extraction
Plant material was thoroughly washed and dried under shade. Dried
material was ground to fine powder. Cold maceration technique was
used for extraction. Powdered plant material (1.5 kg) was dipped in
methanol (200 ml) and kept at room temperature. After 7 days, the
extract was filtered through Whatman filter paper No. 1 under
vacuum. The residue was again dipped in methanol for seven days
and filtered thereafter. The filtrate was combined and the methanol
was evaporated under vacuum using rotary evaporator at 45°C.
The dried extract was stored at 4°C until further analysis.
Fractionation
Four organic and one aqueous fraction were prepared. Fractionation of
crude dry extract was carried out by suspending 250 g of extract in
100 ml water and then partitioned with different organic solvents
(hexane, chloroform, ethyl acetate and methanol) in order of
increasing polarity by using separating funnel (Chiu et al., 2006).
Cell culture
Human breast adenocarcinoma MCF-7 cell line was donated by
Portsmouth University Cell Culture Laboratory, UK. The cells were
maintained in Dulbecco's modified eagle medium (DMEM) supple-
mented with FBS (fetal bovine serum), penicillin (10000 units),
streptomycin (10 mg/ml) and L-glutamine (200 mM) at 37°C under
5% CO2 and relative humidity 95%.
Dilution of extracts
Crude extract and fractions were weighed as needed. Crude/
fractionated extracts were dissolved in dimethyl sulfoxide (DMSO)
at concentration of 2 mg/ml separately. Required dilutions in µg/ml
were made under sterile conditions by adding calculated amounts
of DMEM.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTT assay
Standard MTT assay was used for evaluation of cell viability (Son et
al., 2003). In 96 well plates, MCF-7 cells were seeded at the con-
centration of 5000 cells/well in 100 l medium (RPMI 1640). Cells
were allowed to attach overnight and then various concentrations of
the crude extracts and fractions were added to the wells. After 24 h
incubation, 10 l of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) was added to each well. After 4 h
incubation, 100 l of DMSO solution was added to each well to
solubilize the MTT crystals. The plates were incubated overnight at
37°C, 5% CO2 and relative humidity 95%. The plates were read for
optical density at 570 nm, using a plate reader.
Antibacterial activity
Crude dry extract with a concentration of 20 mg/ml was tested for
antibacterial activity by using agar well diffusion method (Fatima et
al., 2009). Cefotaxime (2 mg/ml) was used as positive control and
pure DMSO was used as a negative control. Bacterial strains used
were Bacillus subtilis, Micrococcus leuteus, Salmonella setubal ,
Salmonella aureus and Pseudomonas picketii. Tests were performed
in triplicates for each microorganism and results were presented as
arithmetic average.
Phytochemical analysis of crude extract and fractions
Different chemical tests were conducted for preliminary phyto-
chemical analysis.
Test for alkaloids
Mayer’s reagent
Mercuric chloride (0.3555 g) was dissolved in 60 ml of water and 5
g of potassium iodide was dissolved in 20 ml of water. Two
solutions were mixed and volume was made up to 1000 ml with
distilled water.
Dragendorff’s reagent
1. Solution A: Basic bismith nitrate (1.7 g) and 20 g of tartaric acid
was dissolved in 80 ml of distilled water.
2. Solution B: Potassium iodide (16 g) was dissolved in 40 ml of
distilled water
Solution A and B were mixed in ratio of 1:1. Plant extract (0.5 - 0.6
g) was mixed with about 8 ml of 1% HCl, warmed and filtered. 2 ml
of filtrate were treated separately with Mayer’s reagent and
Dragendorff’s reagent. Turbidity or precipitation was observed to
indicate the presence of alkaloids.
 Bibi et al. 5613

Table 1. Anticancerous activity of V. foetense crude extract against MCF-7 cell line.

Concentration (µg/ml) Average absorbance Standard deviation Percentage inhibition (%)

10 0.2006 0.002547 28.4
25 0.1505 0.00108 55.6
50 0.1026 0.003921 81.6
100 0.0998 0.01549 83.1
200 0 .0806 0.002221 90.5
300 0.0779 0.001197 92
400 0.0736 0.002171 94
500 0.065 0.001414 98.5

Test for anthraquinones
Plant extract (1 g) was boiled with 6 ml of 1% HCl and filtered. The
filtrate was shaken with 5 ml of benzene. The benzene layer was
removed and then 10% NH4OH was added. Formation of pink,
violet or red color in alkaline phase was observed for the presence
of anthraquinones.
Test for coumarins
Moistened plant extract (0.5 g) was taken in a small test tube and
covered with filter paper moistened with 1 N NaOH. The test tube
was placed for few minutes in boiling water. Then filter paper was
removed and examined in UV light for yellow florescence to indicate
the presence of coumarins.
Test for flavonoids
Prepared extract (0.5 g) was shaken with pet ether to remove the
fatty materials. The defatted residue was dissolved in 20 ml of 80%
of ethanol and filtered. The filtrate was used for the following test:
a) Filtrate (3 ml) was mixed with 4 ml of 1% AlCl3 in MeOH in a test
tube. Formation of yellow color was observed to indicate the
presence of flavonols, flavones and/or chalcones.
b) Filtrate (3 ml) was mixed with 4 ml of 1% KOH. A dark yellow
color was observed to indicate the presence of flavonoids.
Test for saponins
Plant extract (0.5 g) was dissolved in boiling water in a test tube,
allowed to cool and shaken to mix thoroughly. Froth appears
indicated the presence of saponins.
Test for tannins
Plant extract (0.5 g) was boiled in 20 ml of distilled water in a test
tube and then filtered. 0.1% FeCl3 was added to filtrate. Appearance of
brownish green or blue black coloration showed the presence of
tannins.
RESULTS AND DISCUSSION
Plants have been used as traditional medicinal agents
and serve as a base for modern medicines. Crude extract
of V. foetens showed remarkable anticancerous activity
against MCF-7 cell line at all concentrations in a dose
dependent manner (Table 1). The average absorbance
decreased by increasing the concentration of crude
extract. Highest absorbance of 0.2006 was recorded
when determining 28.4% inhibition at 10 µg/ml, while at
the concentration of 500 µg/ml of crude extract, 0.065
absorbance was recorded with 98.5% inhibition. Cordell
(1995) and Kusumoto et al. (1995) also suggested that
crude extracts testing is beneficial for screening of
bioactive components than isolation and testing.
On the basis of significant anticancerous activity of
crude extract against breast cancer cell line MCF-7, the
crude extract was fractionated and fractions at a concen-
tration of 200 µg/ml were again tested against MCF-7 cell
line. Highest percentage inhibition (83%) was measured
by methanol fraction, while chloroform fraction showed
55.5% activity (Figure 1). Hexane fraction demonstrated
25.11% activity, while water fraction showed lowest activity
(2%) against MCF-7 cell line. The maximum inhibition
among fractions at a concentration of 200 µg/ml (83%)
was less than crude extract inhibition at 200 µg/ml
(90.5%). Same findings were reported by several investi-
gations that crude extracts showed more activity than
fractions or separated components (Pellati et al., 2006).
The crude extract of V. foetens was also tested against
some bacterial strains to determine antibacterial potential
of this potent shrub (Table 2). Maximum zone of inhibition
was measured against Salmonella setubal (12.0 ± 0.1),
while mean zone of inhibition 11.2, 11.3 and 11.5 mm
was found against Staphylococcus aureus, Bacillus subtillis
and Micrococcus luteus, respectively. Minimum zone of
inhibition 10.3 mm was measured against Pseudomonas
picketii. Although crude methanol extract of V. foetens
proved strongly cytotoxic yet, it showed moderate anti-
bacterial activity at a concentration of 20 mg/ml. These
results are in agreement with findings of Hayet et al.
(2007) that Salvia sclarea extract showed high cytotoxicity
but moderate antimicrobial activity.
Preliminary phytochemical analysis showed presence
of anthraquinones, saponins, tannins, flavonoids and
coumarins in crude extract (Table 3). While methanol
fraction that showed elevated anticancerous activity among
all fractions was negative for presence of anthraquinones
and saponins. So inhibition of crude extract might be from
5614 Afr. J. Biotechnol.

Figure 1. Anticancerous potential of fractions of V. foetens methanol extract (200 µg/ml).

Table 2. Antibacterial activity of crude V. foetens these phytochemicals either synergistically as suggested
extract. by Choo et al. (2001) that activity of a fraction of plant
extract might be due to synergistic effect of phyto-
Bacterial strains Zone of inhibition (mm) chemical constituents present in the extract sample or
P. picketii 10.3 ± 1 individually. Finally, the present assay-guided findings
S. aureus 11.2 ± 0.5 proved that methanol fraction had cytotoxic potential and
S. satuball 12.0 ± 0.1 can be proceeded for isolation studies in future as done
M. leutus 11.5 ± 0 in Viburnum awabuki in which bioactivity guided phyto-
B. subtillis 11.3 ± 0 chemistry led to the isolation of four cytotoxic compounds
DMSO - from cytotoxic fractions ( El-Gamal, 2008).
Cefotaxime 32 ± 0.2

any of these components. The same is reported by Meyer
et al. (1982) that antiproliferative, antitumor, pesticidal
and other activities in crude extracts might be due to
some active principles.
The role of phytochemicals in bioactivities is well
established (Edeoga et al., 2005). Flavonoids are
considered as potent agents against cancer, microbes
and tumors (Farquar, 1996; Lopez-Lazaro, 2002). Tannins
are also reported to have a strong inhibition of tumors.
Likewise, coumarins and their derivatives are another
class of cytotoxic compounds showing activity in plants
(Cao et al., 1998). In view of strong cytotoxic potential of
flavonoids, tannins and coumarins maximum inhibition of
methanol fraction is strongly suggested to be due to
Conclusions
The present study further support the idea that ethano-
botanically important plants can be promising source of
anticancer agents and methanol fraction of V. foetens is a
good candidate for isolation of anticancerous compounds.
These results will form the basis for selection of this plant
specie for further investigation of novel bioactive com-
ponents and their application in pharmaceutical purposes.
ACKNOWLEDGEMENTS
W e are thankful to Higher Education Commission, Pakistan
for provision of grant for this research work and
Portsmouth University, UK for Providing MCF-7 cell line.

Table 3. Phytochemical analysis of crude extract and fractions.
Crude Hexane Chloroform
Phytochemical
extract fraction fraction
Alkaloid - - - -
Flavonoid +++ + +
Coumarins ++ - -
Anthraquinones ++ - -
Saponins +++ - -
Tannins ++ - -
- = Absent, + = low, ++ = moderate, and +++ = high.
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